Barbara Simionati

Mutation in Human Desmoplakin Domain Binding to Plakoglobin Causes a Dominant Form of Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVD/C) is a genetically heterogeneous disease characterized by progressive degeneration of the right ventricular myocardium and increased risk of sudden death. Here, we report on a genome scan in one Italian family in which the disease appeared unlinked to any of the six different ARVD loci reported so far; we identify a mutation (S299R) in exon 7 of desmoplakin (DSP), which modifies a putative phosphorylation site in the N-terminal domain binding plakoglobin. It is interesting that a nonsense DSP mutation was reported elsewhere in the literature, inherited as a recessive trait and causing a biventricular dilative cardiomyopathy associated with palmoplantar keratoderma and woolly hairs. Therefore, different DSP mutations might produce different clinical phenotypes, with different modes of inheritance.

Do the four clades of the mtDNA haplogroup L2 evolve at different rates

Forty-seven mtDNAs collected in the Dominican Republic and belonging to the African-specific haplogroup L2 were studied by high-resolution RFLP and control-region sequence analyses. Four sets of diagnostic markers that subdivide L2 into four clades (L2a-L2d) were identified, and a survey of published African data sets appears to indicate that these clades encompass all L2 mtDNAs and harbor very different geographic/ethnic distributions. One mtDNA from each of the four clades was completely sequenced by means of a new sequencing protocol that minimizes time and expense. The phylogeny of the L2 complete sequences showed that the two mtDNAs from L2b and L2d seem disproportionately derived, compared with those from L2a and L2c. This result is not consistent with a simple model of neutral evolution with a uniform molecular clock. The pattern of nonsynonymous versus synonymous substitutions hints at a role for selection in the evolution of human mtDNA. Regardless of whether selection is shaping the evolution of modern human mtDNAs, the population screening of L2 mtDNAs for the mutations identified by our complete sequence study should allow the identification of marker motifs of younger age with more restricted geographic distributions, thus providing new clues about African prehistory and the origin and relationships of African ethnic groups.

Haplogroup Effects and Recombination of Mitochondrial DNA: Novel Clues from the Analysis of Leber Hereditary Optic Neuropathy Pedigrees

The mitochondrial DNA (mtDNA) of 87 index cases with Leber hereditary optic neuropathy (LHON) sequentially diagnosed in Italy, including an extremely large Brazilian family of Italian maternal ancestry, was evaluated in detail. Only seven pairs and three triplets of identical haplotypes were observed, attesting that the large majority of the LHON mutations were due to independent mutational events. Assignment of the mutational events into haplogroups confirmed that J1 and J2 play a role in LHON expression but narrowed the association to the subclades J1c and J2b, thus suggesting that two specific combinations of amino acid changes in the cytochrome b are the cause of the mtDNA background effect and that this may occur at the level of the supercomplex formed by respiratory-chain complexes I and III. The families with identical haplotypes were genealogically reinvestigated, which led to the reconnection into extended pedigrees of three pairs of families, including the Brazilian family with its Italian counterpart. The sequencing of entire mtDNA samples from the reconnected families confirmed the genealogical reconstruction but showed that the Brazilian family was heteroplasmic at two control-region positions. The survey of the two sites in 12 of the Brazilian subjects revealed triplasmy in most cases, but there was no evidence of the tetraplasmy that would be expected in the case of mtDNA recombination.

Autosomal Dominant Lateral Temporal Epilepsy: Clinical Spectrum, New Epitempin Mutations, and Genetic Heterogeneity in Seven European Families

Summary:  Purpose: To describe the clinical and genetic findings of seven additional pedigrees with autosomal dominant lateral temporal epilepsy (ADLTE).

Characterization of 16 novel human genes showing high similarity to yeast sequences

The entire set of open reading frames (ORFs) of Saccharomyces cerevisiae has been used to perform systematic similarity searches against nucleic acid and protein databases: with the aim of identifying interesting homologies between yeast and mammalian genes. Many similarities were detected: mostly with known genes. However: several yeast ORFs were only found to match human partial sequence tags: indicating the presence of human transcripts still uncharacterized that have a homologous counterpart in yeast. About 30 such transcripts were further studied and named HUSSY (human sequence similar to yeast). The 16 most interesting are presented in this paper along with their sequencing and mapping data. As expected: most of these genes seem to be involved in basic metabolic and cellular functions (lipoic acid biosynthesis: ribulose‐5‐phosphate‐3‐epimerase: glycosyl transferase: β‐transducin: serine‐threonine‐kinase: ABC proteins: cation transporters). Genes related to RNA maturation were also found (homologues to DIM1: ROK1‐RNA‐elicase and NFS1). Furthermore: five novel human genes were detected (HUSSY‐03: HUSSY‐22: HUSSY‐23: HUSSY‐27: HUSSY‐29) that appear to be homologous to yeast genes whose function is still undetermined. More information on this work can be obtained at the website http://grup.bio.unipd.it/hussy Copyright

Microbial dynamics during shelf-life of industrial Ricotta cheese and identification of a Bacillus strain as a cause of a pink discolouration.

Dairy products are perishable and have to be preserved from spoilage during the food chain to achieve the desired shelf-life. Ricotta is a typical Italian soft dairy food produced by heat coagulation of whey proteins and is considered to be a light and healthy product. The shelf-life of Ricotta could be extended, as required by the international food trade market; however, heat resistant microflora causes spoilage and poses issues regarding the safety of the product. Next-generation sequencing (NGS) applied to the Ricotta samples defined the composition of the microbial community in-depth during the shelf-life. The analysis demonstrated the predominance of spore-forming bacteria throughout the shelf-life, mostly belonging to Bacillus, Paenibacillus and Clostridium genera. A strain involved in spoilage and causing a pink discolouration of Ricotta was isolated and characterised as Bacillus mycoides/weihenstephanensis. This is the first report of a food discolouration caused by a toxigenic strain belonging to the Bacillus cereus group that resulted the predominant strain in the community of the defective ricotta. These results suggest that the processing of raw materials to eliminate spores and residual microflora could be essential for improving the quality and the safety of the product and to extend the shelf-life of industrial Ricotta.

Combining ontologies and workflows to design formal protocols for biological laboratories

BackgroundLaboratory protocols in life sciences tend to be written in natural language, with negative consequences on repeatability, distribution and automation of scientific experiments. Formalization of knowledge is becoming popular in science. In the case of laboratory protocols two levels of formalization are needed: one for the entities and individuals operations involved in protocols and another one for the procedures, which can be manually or automatically executed. This study aims to combine ontologies and workflows for protocol formalization.ResultsA laboratory domain specific ontology and the COW (Combining Ontologies with Workflows) software tool were developed to formalize workflows built on ontologies. A method was specifically set up to support the design of structured protocols for biological laboratory experiments. The workflows were enhanced with ontological concepts taken from the developed domain specific ontology.The experimental protocols represented as workflows are saved in two linked files using two standard interchange languages (i.e. XPDL for workflows and OWL for ontologies). A distribution package of COW including installation procedure, ontology and workflow examples, is freely available from http://www.bmr-genomics.it/farm/cow.ConclusionsUsing COW, a laboratory protocol may be directly defined by wet-lab scientists without writing code, which will keep the resulting protocols specifications clear and easy to read and maintain.

A Multi-Omics Approach to Evaluate the Quality of Milk Whey Used in Ricotta Cheese Production

In the past, milk whey was only a by-product of cheese production, but currently, it has a high commercial value for use in the food industries. However, the regulation of whey management (i.e., storage and hygienic properties) has not been updated, and as a consequence, its microbiological quality is very challenging for food safety. The Next Generation Sequencing (NGS) technique was applied to several whey samples used for Ricotta production to evaluate the microbial community composition in depth using both RNA and DNA as templates for NGS library construction. Whey samples demonstrating a high microbial and aerobic spore load contained mostly Firmicutes; although variable, some samples contained a relevant amount of Gammaproteobacteria. Several lots of whey acquired as raw material for Ricotta production presented defective organoleptic properties. To define the volatile compounds in normal and defective whey samples, a headspace gas chromatography/mass spectrometry (GC/MS) analysis was conducted. The statistical analysis demonstrated that different microbial communities resulted from DNA or cDNA library sequencing, and distinguishable microbiota composed the communities contained in the organoleptic-defective whey samples.

Immediate early genes induced by H-Ras in thyroid cells

Expression of oncogenic v-H-Ras in the thyroid cell line FRTL-5 (FRTL-5Ras) results in uncontrolled proliferation, loss of thyroid-specific gene expression and tumorigenicity. Concomitant expression of constitutively activated MEK and Rac, two major H-Ras downstream effectors, in FRTL-5 (FRTL-5MEK/Rac) recapitulates H-Ras effects on proliferation and morphology. In contrast to FRTL-5Ras, however, FRTL-5MEK/Rac cells remain differentiated and are not tumorigenic. To find H-Ras induced genes potentially responsible for tumorigenicity and loss of differentiation, we have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, between de-differentiated and tumorigenic FRTL-5Ras cells and differentiated and non-tumorigenic FRTL-5MEK/Rac cells. We examined 800 of the cDNA clones obtained after subtraction and verified their levels of expression in the two cell lines by reverse northern, identifying 337 H-Ras induced genes. By sequence analysis, we clustered 57 different genes. Among these, 39 were known genes (involved in diverse signal transduction processes regulating mitogenic activity, cell survival, cytoskeletal reorganization, stress response and invasion) while the remaining 18 clones were novel genes. Among the 57 H-Ras specific clones, we identified those genes whose expression is induced early by H-Ras. We suggest that these immediate-early genes may play a crucial role in H-Ras-mediated transformation in thyroid epithelial cells.

Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes.Marianna Pauletto, Tereza Manousaki et al. present the genome sequence of Sparus aurata, a sequential hermaphroditic fish. Comparative analysis with gonochoristic species shows that sex-biased genes generally evolve more slowly in S. aurata due to stronger functional constraints.